Summary DNA replication stress has been recognized as a major source for the genome instability associated with numerous diseases, including cancer. Therefore, understanding the molecular basis of replication stress and, conversely, how accurate, complete, and rapid chromosomal DNA replication is achieved during normal cell proliferation, is crucial for understanding the mechanisms that maintain or threaten genome stability during normal development and disease, respectively. Here we propose experiments that will illuminate both the intrinsic mechanism of the eukaryotic DNA replication machinery and its response to diverse replication stress conditions. In the previous grant cycle we have generated a fully reconstituted origin-dependent DNA replication system based on purified budding yeast proteins. This system forms the central platform for research in our lab over the next funding cycle. In Aim 1 we will exploit the unique biochemical tractability of this system to elucidate the mechanism of the eukaryotic replicative DNA helicase, CMG (Cdc45-Mcm2-7-GINS), which is at the center of the replisome. The CMG is unique among replicative DNA helicases from the three domains of life in that its helicase motor, the Mcm2-7 complex, is composed of 6 distinct subunits. The reasons for this complexity are still obscure. Intriguingly, we have identified a previously unrecognized essential role for the unique unstructured N-terminal tail of Mcm2. Our preliminary data indicate that this domain is involved in multiple functions at the replisome, including DNA unwinding, priming, and chromatin replication. We propose to characterize the unexpected functional versatility of this helicase domain as a gateway to the elucidation of the intrinsic mechanism of a eukaryotic replisome. The focus of Aims 2 and 3 will be to expand the basic DNA replication system for the study of DNA replication stress mechanisms. Experiments in Aim2 build on our original reconstitution of unidirectional replisome collisions with R-loops, a co-transcriptionally formed nucleic acid structure that has been recognized to pose a major threat to genome stability in all organisms. We find that R-loops form an intrinsic barrier to replisome progression by creating a physical block to the fork. A large number of proteins, such as helicases and RNase H-type nucleases, have been implicated in promoting DNA replication in vivo by resolving R-loops. However, the mechanisms are largely unknown. We have purified these accessory enzymes and will characterize their role in promoting fork progression through R-loops. The basis for Aim 3 is our observation that dNTP depletion, one of the most-studied forms of replication stress, causes uncoupling of replisome progression from DNA synthesis in the reconstituted system. We use this reaction to investigate the mechanism by which the checkpoint regulates stalled fork stability, focusing on the central checkpoint effector kinase, Rad53.